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. 2020 Feb 5:179:113002.
doi: 10.1016/j.jpba.2019.113002. Epub 2019 Nov 20.

Development and validation of a volumetric absorptive microsampling- liquid chromatography mass spectrometry method for the analysis of cefepime in human whole blood: Application to pediatric pharmacokinetic study

Ganesh S Moorthy  1 , Christina Vedar  2 , Nicole R Zane  2 , Kevin J Downes  3 , Janice L Prodell  4 , Mary Ann DiLiberto  4 , Athena F Zuppa  4
Affiliations

Development and validation of a volumetric absorptive microsampling- liquid chromatography mass spectrometry method for the analysis of cefepime in human whole blood: Application to pediatric pharmacokinetic study

Ganesh S Moorthy et al. J Pharm Biomed Anal. .

Abstract

Cefepime is a fourth-generation cephalosporin antibiotic with an extended spectrum of activity against many Gram-positive and Gram-negative bacteria. There is a growing need to develop sensitive, small volume assays, along with less invasive sample collection to facilitate pediatric pharmacokinetic clinical trials and therapeutic drug monitoring. The volumetric absorptive microsampling (VAMS™) approach provides an accurate and precise collection of a fixed volume of blood (10 μL), reducing or eliminating the volumetric blood hematocrit assay-bias associated with the dried blood spotting technique. We developed a high-performance liquid chromatographic method with tandem mass spectrometry detection for quantification of cefepime. Sample extraction from VAMS™ devices, followed by reversed-phase chromatographic separation and selective detection using tandem mass spectrometry with a 4 min runtime per sample was employed. Standard curves were linear between 0.1-100 μg/mL for cefepime. Intra- and inter-day accuracies were within 95.4-113% and precision (CV) was < 15 % based on a 3-day validation study. Recoveries ranged from 40.8 to 62.1% and the matrix effect was within 89.5-96.7% for cefepime. Cefepime was stable in human whole blood under assay conditions (3 h at room temperature, 24 h in autosampler post-extraction). Cefepime was also stable for at least 1 week (7 days) at 4 °C, 1 month (39 days) at -20 °C and 3 months (91 days) at -78 °C as dried microsamples. This assay provides an efficient quantitation of cefepime and was successfully implemented for the analysis of whole blood microsamples in a pediatric clinical trial.

Keywords: Cefepime; Human whole blood; Liquid chromatography; Mass spectrometry; Volumetric absorptive microsampling (VAMS™).

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Conflict of interest statement

Conflict of interest: Authors declare that there is no conflict of interest regarding the publication of this paper.

Figures

Figure 1.
Figure 1.
Schematic of sample preparation for cefepime VAMS™-LC-MS/MS assay.
Figure 2.
Figure 2.
Stability of cefepime (10 μg/mL) in PBS, plasma, blood, DBS and VAMS™ at room temperature. Expanded graph from 0 – 3 h is shown as an insert.
Figure 3.
Figure 3.
Chromatograms for cefepime (left), and internal standard cefepime-d3 (right) in human whole blood using VAMS™ for A) double blank, B) single blank and c) LLOQ (0.100 μg/mL).
Figure 4.
Figure 4.
Pharmacokinetic profile of cefepime in a subject (11 kg) following intravenous infusion of cefepime over 30 minutes (50 mg/kg).
See this image and copyright information in PMC

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